Cross-linking of pyruvate decarboxylase. Characterization of the native and substrate-activated enzyme states.
In order to demonstrate the role of the protein component of pyruvate decarboxylase in the mechanism of substrate activation, we have isolated and characterized two states of the enzyme, the non-activated and the substrate-activated state, by covalent linking with bifunctional reagents. Because of the fact that modification of the reactive amino groups by 2,4,6-trinitobenzenesulfonic acid or methyl propionimidate influences neither the catalytic nor the regulatory properties of pyruvate decarboxylase, we used bisimidates of different chain length in the modification experiments. Both the non-activated and the substrate-activated enzyme states could be characterized separately. The lag phase of product formation as a typical property of the native enzyme disappeared completely when the enzyme had been cross-linked in the presence of the substrate. The permanently activated enzyme state shows 85% of the activity of native pyruvate decarboxylase and is exclusively stabilized by intra-subunit links. Elimination and subsequent reincorporation of the cofactors thiamine pyrophosphate and magnesium ions resulted in a complete regaining of the properties of the permanently activated enzyme form. An inactive enzyme form was obtained after cross-linking of non-activated pyruvate decarboxylase at low ionic strength (less than 0.01). Using a disulfide-containing linker we could prove that the inactivity of the obtained enzyme preparation was only the result of the incorporated cross-links and not that of denaturation.
König
S
S
Biotechnikum, Martin-Luther-Universität Halle-Wittenberg, GDR.
Hübner
G
G
Schellenberger
A
A
D003432:Cross-Linking Reagents; D004789:Enzyme Activation; D007700:Kinetics; D015394:Molecular Structure; D011487:Protein Conformation; D011767:Pyruvate Decarboxylase / Q000737:chemistry / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000737:chemistry
D003432:Cross-Linking Reagents; D013438:Sulfhydryl Compounds; D011767:Pyruvate Decarboxylase